The invention relates to nucleic acids and protein of the type 3 human ryanodine receptor (hRyR3), chimeric ryanodine receptors with parts of the human receptor and processes for preparing these proteins. The invention further relates to the detection of ryanodine receptors in human tissues for diagnosing pathological conditions and methods of identifying activators or inhibitors of hRyR3.
Cytoplasmic calcium plays an important part in cell activation, the release of neurotransmitters, muscle contraction and other biological processes. It is increased by the effect of extracellular calcium resulting from voltage-activated and other ion channels and by the calcium release from intracellular supplies. At present, two intracellular calcium release channels are known, the inositol 1,4,5-triphosphate receptors (IP3R) and the ryanodine receptors (RyR). The release of calcium by IP3R starts from a ubiquitous mechanism which has been described for numerous cells. By contrast, three types of RyR-mRNA, namely RyR1, RyR2 and RyR3 are expressed tissue-specifically; RyR1 primarily in the skeletal muscle, RyR2 in the heart muscle and brain and RyR3 in the brain and smooth muscle. In the brain, the RyR3 is strongly expressed only in very limited areas such as the hippocampus, Nucleus caudatus, Corpus callosum and thalamus. The RyR3 is also expressed in non-excitable cells such as human T-lymphocytes. It has been postulated that RyR3 has a part to play in cell proliferation (Hakamata, Y. et al. FEBS Lett., 352 (1994), 206-210). For RyR1 and RyR2 it has been shown that, in the excitation contraction coupling of skeletal and heart muscle, voltage-activated calcium channels activate the RyR1 in the skeletal muscle and presumably also in neurones directly, whereas the calcium of the voltage-activated channels is a trigger for the opening of RyR2 in heart muscle (calcium-induced release of calcium). The function of RyR3 is subject to a series of speculations. Although calcium appears to be an important physiological ligand of RyR3, there are some indications that the calcium-induced release of calcium differs from that of other RyR. It is assumed that an endogenous RyR3 is responsible for the substantially lower calcium sensitivity of the remaining calcium release activity of RyR1-deficient murine muscle cells. RyR3 is demonstrably insensitive to caffeine in some cases, caffeine being the substance primarily used for RyR activation. Since RyR3 is expressed in non-excitable cells which have virtually no voltage activated calcium channels, it appears possible that RyR3 is regulated by different mechanism from the other RyR. RyR3-deficient mutant mice exhibit increased locomotor activity. The cDNA sequences of RyR1, RyR2 and for rabbit-RyR3 (rRyR3) are already known whereas the nucleic acid sequence of RyR3 in humans (hRyR3) has not yet been investigated.
In spite of a plethora of bits of information regarding RyR3, its molecular physiological properties, its significance in pathological conditions and methods of evaluating possible inhibitors and activators of its activity are substantially or even totally unknown. In addition, the transfer of the currently available information from tests with isolated RyR3 of non-human origin to humans is accompanied by considerable uncertainty.